Novel Fusion Protein Comprising Transcription Modulation Domain of p65 and Protein Transduction Domain, and Uses Thereof

ABSTRACT

The present invention relates to a novel fusion protein comprising the transcription modulation domain of the transcription factor NF-κB subunit p65 and a protein transduction domain and to the use thereof. The fusion protein of the present invention has the effects of inhibiting the transcription of NF-κB and IL-2 by competitive inhibition and inhibiting the LPS-induced secretion of inflammatory cytokines and also inhibiting the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 in splenocytes, and thus is effectively used as a composition for the prevention or treatment of a disease related to NF-κB overactivity.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a divisional of U.S. Ser. No. 15/342,264, filed onNov. 3, 2016, which claims priority to Korean Patent Application No.10-2015-0142144, filed on Oct. 10, 2015, the disclosures of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a novel fusion protein comprising thetranscription modulation domain (TMD) of the transcription factor NF-κBsubunit p65 and a protein transduction domain (PTD) and to the usethereof.

Description of the Related Art

Cytokines that are regulated by the transcription factor NF-κB includetumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), interleukin-6 andgranulocyte-macrophage colony stimulating factor (GM-CSF), andchemokines that are regulated by NF-κB include interleukin-8,macrophage-inflammatory protein-1α (MIP-1α), methyl accepting chemotaxisprotein-1 (MCP-1) and eotaxin. In addition, adhesion molecules that areregulated by NF-κB include E-selectin, vascular cell adhesion molecule-1(VCAM-1), endothelial leukocyte adhesion molecule-1 (ELAM-1) andintercellular cell adhesion molecule-1 (ICAM-1), and inducible enzymesthat are regulated by NF-κB include cyclooxygenase-2 (COX-2) and thelike. Thus, NF-κB is involved in almost all physiological reactions inthe body.

The transcription factor NF-κB is composed of different subunitscomposed of homodimers or heterodimers. NF-κB proteins include RelA(p65), c-Rel, Rel-B, NF-κB1 (p50) and NF-κB2 (p52), and p50 and p52 areproduced from NF-κB1 (p105) and NF-κB2 (p100), respectively, which aretheir precursors. The NF-κB proteins each contains about 300 aminoacids, called the N-terminal Rel-homology domain (RHD), which isinvolved in dimerization, binding to specific DNA and reactions with IκBprotein. Furthermore, these proteins also contain a nuclear localizationsignal (NLS) that enables the proteins to act as transcription factorsby localization to the nucleus. In addition, the NF-κB proteins may beclassified, according to the presence or absence of the C-terminaltransactivation domain (TAD), into class I (NF-κB1 and NF-κB2) and classII (RelA/p65, RelB, and c-Rel). Class II has a transactivation domainthat enables the NF-κB proteins to act as transcription factors withoutneeding other NF-κB domains, and class I has no transactivation domainDimers are formed between the NF-κB proteins of the two classes to actas DNA transcription factors. Among the dimers, a p50/p65 dimer is mostfrequently present. RelA(p65), RelB and c-Rel have a C-terminaltransactivation domain, and thus can activate the expression of theirtarget genes. On the contrary, p50 and p52, which are NF-κB1 and NF-κB2,respectively, have no C-terminal transactivation domain, and thushomodimers of p50 and p52 do not act as transcription factors if they donot have bound thereto proteins such as co-activators having atransactivation domain.

A protein transduction domain (PTD) is a short peptide having stronghydrophobicity, and is known to effectively transduce physiologicallyactive molecules such as proteins, DNA and RNA, fused therewith, intocells. The present inventors have developed two PTDs to date, and thePTDs are disclosed in detail in WO 2003059940 and WO 2003059941. Becausethe protein transduction domain can transduce a physiologically activemolecule not only into the cytoplasm but also into the nucleus, it has aproperty suitable for transducing a modified transcription factor, whichis the key substance of the present invention, into the nucleus.

Therefore, the present inventors have attempted to use a fusion proteincomprising the transduction modulation domain of the NF-κB subunit p65and a protein transduction domain to inhibit the transcription andactivity of NF-κB by competitive inhibition to thereby treat effectivelya disease caused by NF-κB overactivity.

Throughout the specification, a number of publications and patentdocuments are referred to and cited. The disclosure of the citedpublications and patent documents is incorporated herein by reference inits entirety to more clearly describe the state of the related art andthe present invention.

SUMMARY OF THE INVENTION

The present inventors have made extensive efforts to develop a substancecapable of effectively preventing, alleviating or treating a diseasecaused by the overactivity of NF-κB. As a result, the present inventorshave found that, when a fusion protein comprising the transcriptionmodulation domain of the NF-κB subunit p65 and a protein transductiondomain is used, it can inhibit the transcription and activity of NF-κBby competitive inhibition, thereby completing the present invention.

Therefore, it is an object of the present invention to provide a fusionprotein comprising the transcription modulation domain of the NF-κBsubunit p65 and a protein transduction domain.

Another object of the present invention is to provide an inhibitor ofNF-κB transcription or activity, comprising the fusion protein of thepresent invention.

Still another object of the present invention is to provide apharmaceutical composition for the prevention or treatment of a diseaserelated to NF-κB overactivity, the pharmaceutical composition comprisingthe fusion protein of the present invention as an active ingredient.

Other objects and advantages of the present invention will become moreapparent from the following detailed description, the appended claimsand the accompanying drawings.

In accordance with one aspect of the present invention, there isprovided a fusion protein comprising the transcription modulation domainof the NF-κB subunit p65 and a protein transduction domain. The fusionprotein of the present invention inhibits NF-κB transcription bycompetitive inhibition.

The present inventors have made extensive efforts to develop a substancecapable of effectively preventing, alleviating or treating a diseasecaused by NF-κB overactivity. As a result, the present inventors havefound that, when a fusion protein comprising the transcriptionmodulation domain of the NF-κB subunit p65 and a protein transductiondomain is used, it can inhibit the transcription and activity of NF-κBby competitive inhibition.

As used herein, the term “transcription modulation domain” means aportion of a transcription factor, which is composed only of a DNAbinding domain without a transactivation domain. As demonstrated in theexamples below, the fusion protein of the present invention has notransactivation domain, but has a DNA binding domain, and thus it canbind to a target promoter, but cannot promote transcription.Accordingly, the fusion protein of the present invention is a dominantnegative mutant for the NF-κB gene, and thus can act as a competitiveinhibitor against wild-type NF-κB in cells to inhibit the transcriptionand activity of NF-κB.

In one embodiment, NF-κB that is used in the present invention is NF-κBselected from the group consisting of RelA (p65), c-Rel, Rel-B, NF-κB1(p50) and NF-κB2 (p52). In a specific embodiment, NF-κB that is used inthe present invention is RelA (p65).

In one embodiment, the transcription modulation domain of NF-κB that isused in the present invention comprises an amino acid sequence of SEQ IDNO: 1. In another embodiment of the present invention, the transcriptionmodulation domain of NF-κB, which comprises the amino acid sequence ofSEQ ID NO: 1, is encoded by a nucleotide sequence of SEQ ID NO: 3.

As used herein, the term “protein transduction domain” is a short,strongly hydrophobic peptide consisting of 7-50 amino acids, and means adomain capable of transducing not only a protein having a molecularweight of 120 kDa or more, but also DNA or RNA, into cells. Asdemonstrated in the examples below, it could be seen that, unlike thefusion protein of the present invention, a protein to which the proteintransduction domain of the present invention is not attached (that is,p65-TMD composed only of the transcription modulation domain) has noeffects on the inhibition of transcription of NF-κB and IL-2, theinhibition of LPS-induced secretion of inflammatory cytokines, and theinhibition of production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 insplenocytes.

According to one embodiment of the present invention, the proteintransduction domain that is used in the present invention is selectedfrom the group consisting of Hph-1, Mph-1, Sim-2, Tat, VP22, Antp(antennapedia), Pep-1 (peptide-1), PTD-5 (protein transductiondomain-5), 11R, 7R and CTP (cytoplamic transduction peptide). As usedherein, the terms “11R” and “7R” mean peptides consisting of 11 and 7arginine residues, respectively. According to a specific embodiment, theprotein transduction domain that is used in the present invention isHph-1.

According to one embodiment of the present invention, the proteintransduction domain that is used in the present invention comprises anamino acid sequence of SEQ ID NO: 2. According to another embodiment ofthe present invention, the protein transduction domain comprising theamino acid sequence of SEQ ID NO: 2 according to the present inventionis encoded by a nucleotide sequence of SEQ ID NO: 4.

According to one embodiment of the present invention, the fusion proteinof the present invention comprises an amino acid sequence of SEQ ID NO:5. According to a specific embodiment of the present invention, a fusionprotein comprising an amino acid sequence of SEQ ID NO: 5 is encoded bya nucleotide sequence of SEQ ID NO: 6.

In accordance with another aspect of the present invention, there isprovided an inhibitor of NF-κB transcription or activity, comprising thefusion protein of the present invention. As demonstrated in the examplesbelow, it was shown that the fusion protein of the present inventioncould inhibit the transcription and activity of NF-κB and IL-2 (FIG. 6),inhibit the LPS-induced secretion of inflammatory cytokines (FIG. 7),and inhibit the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 whichare expressed by T-cell activation stimulated by anti-CD3/CD28 insplenocytes (FIG. 9), indicating that the fusion protein can inhibit theNF-κB-induced proliferation and differentiation of T cells and theNF-κB-induced secretion of inflammatory cytokines. This suggests thatthe fusion protein of the present invention can effectively prevent,alleviate or treat a disease caused by the overactivity of NF-κB.

In accordance with another aspect of the present invention, there isprovided a pharmaceutical composition for the prevention or treatment ofa disease related to NF-κB overactivity, the pharmaceutical compositioncomprising the fusion protein of the present invention as an activeingredient.

As demonstrated in the examples below, the fusion protein of the presentinvention inhibits the transcription of NF-κB and IL-2, inhibits theLPS-induced secretion of inflammatory cytokines, and inhibits theproduction of IL-2, IFN-γ, IL-4, IL-17A and IL-10 in splenocytes. Thus,the fusion protein of the present invention may be effectively used as acomposition for efficiently preventing or treating various diseasesrelated to NF-κB overactivity.

In one embodiment of the present invention, the disease related to NF-κBoveractivity in the present invention is an inflammatory disease or anautoimmune disease. In another embodiment of the present invention, thedisease related to NF-κB overactivity in the present invention is adisease selected from the group consisting of septic shock, allergicasthma, allergic nasitis, atopic dermatitis, systemic lupuserythematosus, rheumatoid arthritis, ulcerative colitis, dacryoadenitis,Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, typeI diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis,systemic inflammatory response syndrome, polymyositis, dermatomyositis,polyarthritis nodosa, mixed connective tissue disease, Sjogren'ssyndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis,diabetic retinopathy, multiple sclerosis, Crohn's disease, chronicthyroiditis, Celiac disease, myasthenia gravis, pemphigus vulgaris,viral diseases, bacterial diseases, radiation-induced disorders,arteriosclerosis, hemangioma, angiofibroma, reperfusion injury, andcardiac hypertrophy. According to a specific embodiment, the diseaserelated to NF-κB overactivity in the present invention is septic shock.Septic shock that causes a severe systemic inflammatory reaction due tomicrobial infection shows abnormalities in the cardiovascular system andvasomotor factors by endotoxins and inflammatory mediators circulatingthe blood vessels. For this reason, hypovolemia occurs due todehydration and leakage of intravascular liquid, and vascularcontraction and relaxation in capillary vessels occur to causeabnormalities in blood supply, and vasculitis and thrombosis make tissuereflux more difficult, resulting in tissue hypoxia and metabolicacidosis. In particular, endotoxins activate NF-κB in immunocytes(macrophages, granulocytes, dendritic cells, and lymphocytes) to secretecytokines such as tumor necrosis factor, linterleukin-1 andlinterleukin-6, and such cytokines stimulate neutrophils, endothelialcells, platelets or inflammatory mediator-releasing cells to causesystemic reactions. As demonstrated in the examples below, it was shownthat the fusion protein of the present invention inhibited the secretionof inflammatory cytokines in septic shock models induced by LPS andincreased the survival rate of the models (FIG. 10).

Thus, it can be seen that the fusion protein of the present invention iseffective for the prevention or treatment of a disease related to NF-κBoveractivity.

If the composition of the present invention is prepared as apharmaceutical composition, the pharmaceutical composition of thepresent invention contains a pharmaceutically acceptable carrier.Examples of the pharmaceutically acceptable carrier that is contained inthe pharmaceutical composition of the present invention include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch,acacia gum, calcium phosphate, alginate, gelatin, calcium silicate,microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water,syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate,talc, magnesium stearate, and mineral oil, which are commonly used informulations. The pharmaceutical composition may further contain, inaddition to the above-described components, a lubricant, a wettingagent, a sweetening agent, a flavoring agent, an emulsifying agent, asuspending agent, a preservative or the like. Suitable pharmaceuticallyacceptable carriers and formulations are described in detail inRemington's Pharmaceutical Sciences (19^(th) ed., 1995).

The pharmaceutical composition of the present invention may beadministered orally or parenterally. When the pharmaceutical compositionis administered parenterally, it may be injected intravenously,subcutaneously, intramuscularly, intraperitoneally, transdermally, orthe like. In one embodiment of the present invention, the pharmaceuticalcomposition of the present invention may be administered byintraperitoneal injection.

The suitable dose of the pharmaceutical composition of the presentinvention may vary depending on various factors, including theformulation method, the mode of administration, the patient's age,weight, sex, disease condition and diet, the time of administration, theroute of administration, the rate of excretion, and reactionsensitivity. The daily dose of the pharmaceutical composition accordingto the present invention is, for example, 0.0001-1000 mg/kg.

The pharmaceutical composition of the present invention may beformulated with a pharmaceutically acceptable carrier and/or excipientin a unit dosage form or a multiple dosage form, according to a methodthat can be easily carried out by a skilled person in the art to whichthe present invention pertains. Herein, the formulation may be asolution in oil or an aqueous medium, a suspension, syrup, anemulsifying solution, an extract, powder, granules, a tablet, or acapsule, and may further contain a dispersing agent or a stabilizingagent.

Because the NF-κB transcription or activity inhibitor of the presentinvention and the composition for the prevention or treatment of adisease related to NF-κB overactivity according to the present inventioncommonly comprise the above-described fusion protein, the description ofthe contents that are common in relation to the fusion protein isomitted in order to avoid excessive complexity of description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a fusion protein (nt-p65-TMD) of ap65-transcription modulation domain (p65-TMD) and Hph-1 (PTD) accordingto an embodiment of the present invention. DBD: DNA binding domain; IBD:IκB binding domain; NLS: nuclear localization sequence; TAD:transactivation domain.

FIG. 2 shows the results of Coomassie blue staining of the fusionprotein (nt-p65-TMD) according to the present invention.

FIG. 3a and FIG. 3b show the results of transduction of the fusionprotein (nt-p65-TMD) of the present invention into BV2 and Jurkat Tcells.

FIG. 4 shows the results of transduction of the fusion protein(nt-p65-TMD) of the present invention into the nuclei of BV2 and HeLacells.

FIG. 5 panels A and B show the results of cytotoxicity of the fusionprotein (nt-p65-TMD) of the present invention in BV2 and spleen cells.

FIG. 6 panels A and B show the effect of the fusion protein (nt-p65-TMD)of the present invention on competitive inhibition of transcription.

FIG. 7 panels A, B, and C show the effect of the fusion protein(nt-p65-TMD) of the present invention on the inhibition of LPS-inducedexpression of TNF-α, IL-1β and IL-6.

FIG. 8a shows results indicating that the fusion protein (nt-p65-TMD) ofthe present invention specifically inhibits NF-κB transcription inducedby T-cell activation stimulated by anti-CD3/CD28. FIG. 8b show resultsindicating that fusion protein (nt-p65-TMD) of the present inventiondoes not influence signal transduction pathways induced by T-cellactivation stimulated by anti-CD3/CD28.

FIG. 9 panels A, B, C, D, E, and F show results indicating that thefusion protein (nt-p65-TMD) of the present invention inhibits theexpression of IL-2, IFN-γ, IL-4, IL-17A and IL-10, induced by T-cellactivation stimulated by anti-CD3/CD28.

FIG. 10 panels A, B, C, and D show results indicating that the fusionprotein (nt-p65-TMD) of the present invention inhibits the secretion ofinflammatory cytokines (TNF-α, IL-1β and IL-6) in septic shock animalmodels to thereby increase the survival rate of the animal models.

DESCRIPTION OF SPECIFIC EMBODIMENTS

Hereinafter, the present invention will be described in further detailwith reference to examples. It will be obvious to those skilled in theart that these examples are for illustrative purposes and are notintended to limit the scope of the present invention.

EXAMPLES Example 1: Preparation of a Recombinant Fusion ProteinComprising p65 Transcription Modulation Domain and Protein TransductionDomain

The protein transduction domain (PTD) Hph-1 (SEQ ID NO: 4) was clonedinto the p65 transcription modulation domain p65-TMD (SEQ ID NO: 2) anda pET-28a(+) vector (Novagen) to thereby construct a recombinant fusionDNA (nt-p65-TMD) (SEQ ID NO: 6). The recombinant fusion DNA wastransformed into a BL21 CodonPlus(DE3)-RIPL E. coli strain (Invitrogen).The transformed strain was cultured, and then treated with 1 mM IPTG(isopropyl-β-D-thiogalactopyranoside, Duchefa), after which proteinexpression in the strain was induced at 37° C. for 5 hours. Next, onlythe cells were harvested, lysed with lysis buffer (10 mM imidazole, 50mM NaH₂PO₄, 300 mM NaCl, pH 8.0), and then disrupted with a homogenizer.The fusion protein was bound to Ni-NTA beads (Qiagen) by 6 histidineresidues linked to the upstream region of the protein. The protein wasloaded onto HisTrap chromatography columns (Bio-Rad), and washedsufficiently with washing buffer (30 mM imidazole, 50 mM NaH₂PO₄, 300 mMNaCl, pH 8.0), and the protein was eluted with elution buffer (250 mMimidazole, 50 mM NaH₂PO₄, 300 mM NaCl and pH 8.0). Using PD-10 SephadexG-25 (GE Healthcare), imidazole and NaCl were removed while the bufferwas replaced with 10% glycerol-containing PBS. Because the obtainedprotein contained endotoxins such as LPS, it was purified once moreusing SP beads (SP Sepharose™ Fast Flow, GE Healthcare) to remove theendotoxins. The resulting protein was bound using binding buffer (50 mMNaH₂PO₄, 300 mM NaCl, pH 6.0), and then loaded onto a column and elutedwith elution buffer (50 mM NaH₂PO₄, 2 M NaCl, pH 6.0). Finally, NaCl wasremoved using PD-10 Sephadex G-25, and the buffer was placed with 10%glycerol-containing PBS, after which the resulting protein (SEQ ID NO:5, recombinant fusion protein) was stored at 80° C. until use inexperiments (see FIGS. 1 and 2).

Example 2: Analysis of Intracellular Transduction of nt-p65-TMDRecombinant Fusion Protein

2-1: Analysis of Intracellular Transduction by Western Blotting

BV2 microglial cells and Jurkat T cells were incubated with therecombinant fusion protein (nt-p65-TMD) at varying proteinconcentrations (0, 0.1, 0.5, 1, 2 and 5 μM) or for varying times (0, 1,2, 4, 6, 12, 24 and 48 hours), and whether or not the fusion protein wastransduced was analyzed by Western blotting.

As a result, it was shown that the fusion protein was well transduced inproportion to the concentration thereof and was continuously transducedeven at 48 hours while the protein in the cell culture maintained itsstructure (see FIG. 3).

2-2: Analysis of Whether or not Fusion Protein is Transduced to Nucleusof Cells, by Use of Antibody

5 μM of the recombinant fusion protein (nt-p65-TMD) of Example 1 wasincubated with BV2 microglial cells and HeLa cells for 1 hour and washedwith PBS, after which the cells were treated with 0.2% Triton X-100(Sigma-Aldrich) to form an opening, and a fluorescence-labeled antibodywas bound to the recombinant fusion protein through the opening. Next,the nucleus of the cells was stained with DAPI dye (Invitrogen), andthen the location of fluorescence was determined by a fluorescencemicroscope to thereby determine the location to which the recombinantfusion protein was transduced.

As a result, it was shown that the fusion protein was well transduced tothe nucleus of the cells by the property of the PTD (see FIG. 4).

Example 3: Analysis of Cytotoxicity of nt-p65-TMD Recombinant FusionProtein

In order to confirm that the protein obtained from the E. coli strain byexpression is completely free of LPS so that it is not toxic to cells oranimals, a cytotoxicity test was performed. Varying concentrations ofthe protein were transduced into BV2 microglial cells and splenocytes,and then the cells were incubated with WST-8, a substrate that developscolor by dehydrogenase present in living cells.

As a result, it could be seen that the cells treated with the fusionprotein showed no cytotoxicity regardless of the concentration of thefusion protein, unlike cells not treated with the protein (FIG. 5).

Example 4: Confirmation of the Effect of nt-p65-TMD Recombinant FusionProtein on Specific Inhibition of Transcription Factor

4-1: Confirmation of Inhibitory Effect against Transcription of NF-κBand IL-2 in HEK293T Cells

To directly confirm whether or not the nt-p65-TMD recombinant fusionprotein of Example 1 binds to the promoters of the NF-κB and IL-2cytokine genes in place of wild-type p65 to inhibit the expression ofthe genes, luciferase reporter gene was used. First, each of NF-κB andIL-2 promoters and wild-type p65 gene, which have luciferase in thedownstream region, was transfected into the nucleus of HEK293T cells,and then the cells were treated with the nt-p65-TMD recombinant fusionprotein.

As a result, it could be seen that, when the cells were treated with therecombinant fusion protein, the expression of luciferase in the cellswas effectively inhibited. This suggests that nt-p65-TMD acted as acompetitive inhibitor of wild-type p65 to block the p65-binding site ofeach of the NF-κB and IL-2 promoters (see FIG. 6).

4-2: Confirmation of Inhibitory Effect Against Secretion of InflammatoryCytokines in BV2 Microglial Cells

BV2 microglial cells were treated with the nt-p65-TMD recombinant fusionprotein of Example 1, and after 1 hour, the cells were treated andincubated with LPS (1 μg/ml, E. coli serotype 055:B5, Sigma-Aldrich) for24 hours.

As a result, it could be seen that the expression of TNF-α, IL-1β andIL-6, which are activated and expressed by LPS, was inhibited by thent-p65-TMD recombinant fusion protein (see FIG. 7).

4-3: Confirmation of Specific Inhibitory Effect Against NF-κBTranscription Factor in Jurkat T Cells

Whether or not the nt-p65-TMD recombinant fusion protein of Example 1specifically inhibits the transcription of NF-κB activated by JurkatT-cell activation stimulated by anti-CD3 (1 μg/ml, BD Pharmingen) andanti-CD28 (1 μg/ml, BD Pharmingen) was examined. When T cells areactivated, not only NF-κB transcription but also NFAT transcription isactivated. For this reason, if the nt-p65-TMD fusion protein inhibitsonly NF-κB transcription without influencing NFAT transcription, it canbe seen that the nt-p65-TMD recombinant fusion protein acts as aspecific competitive inhibitor of NF-κB. To confirm this fact,luciferase reporter gene was used. First, NF-κB and NFAT reporter geneshaving luciferase in the downstream region were transfected into thenucleus of Jurkat T cells by electroporation, and then the Jurkat Tcells were stimulated with anti-CD3 and anti-CD28 and treated with thent-p65-TMD recombinant fusion protein.

As a result, it could be seen that, when the NF-κB-transfected cellswere treated with the recombinant fusion protein, the expression ofluciferase in the cells was effectively inhibited, but NFAT was notinfluenced by the recombinant fusion protein (see FIG. 8a ).

4-4: Phosphorylation of Intracellular Signal Transduction Protein

In order to examine whether or not the nt-p65-TMD recombinant fusionprotein of Example 1 is involved in tyrosine phosphorylation of proteinsrelated to various intracellular signal transduction systems, Westernblot analysis was performed. Jurkat T cells were treated with 2 μM ofnt-p65-TMD for 1 hour, and then stimulated with anti-CD3 (2.5 μg/ml) andanti-CD28 (2.5 μg/ml), and whether or not tyrosine phosphorylation ofZAP-70, p38, JNK or ERK occurred was observed.

As a result, it could be seen that nt-p65-TMD did not influencephosphorylation of these proteins (see FIG. 8b ).

4-5: Inhibition of Production of Cytokines (IL-2, IFN-γ, IL-4, IL-17Aand IL-10) in Splenocytes

Splenocytes isolated from the spleens of 6-8-week-old female C57BL/6mice were treated with the nt-p65-TMD recombinant fusion protein ofExample 1 for 1 hour to transduce the recombinant fusion protein intothe cells. The cells were stimulated with anti-CD3 (1 μg/ml) andanti-CD28 (1 μg/ml), and then incubated for 72 hours. Next, the amountsof cytokines present in the medium were measured by ELISA.

As a result, it could be seen that, when the cells were treated withnt-p65-TMD, the expression levels of IL-2, IFN-γ, IL-4, IL-17A and IL-10in the cells were greatly inhibited. In addition, the expression levelof CD69, which is indicative of T cell activation, was measured by FACSCalibur (BD Biosciences), and as a result, it could be seen thatnt-p65-TMD did not influence the expression of CD69 in T cells (FIG. 9).

Example 5: Evaluation of Therapeutic Effect of nt-p65-TMD in SepticShock Animal Models

LPS (20 mg/kg) was injected intraperitoneally into 6-8-week-old maleBALB/c mice to make septic shock animal models. After 2 hours and 14hours, the nt-p65-TMD recombinant fusion protein of Example 1 wasinjected intraperitoneally into the animal models, followed byobservation for 6 days.

As a result, it could be seen that, when the animal models were treatedwith nt-p65-TMD, the secretion of inflammatory cytokines (TNF-α, IL-1βand IL-6) was inhibited to thereby greatly increase the survival rate ofthe animal models (see FIG. 10).

Example 6: Acute Toxicity Test for Recombinant Fusion Protein

Using 6-week-old specific pathogen-free (SPF) rats obtained from theDaehan Experiment Supply Center, an acute toxicity test was performed inthe following manner. The recombinant fusion protein of Example 1 wasadministered orally once to each animal group (consisting of twoanimals) at a dose of 1 g/kg, and then the death, clinical symptoms andweight changes of the animals were observed, and hematological tests andblood biochemical tests were performed. In addition, the animals wereautopsied, and whether or not the abdominal organs and the thoracicorgans were abnormal was visually observed.

As a result, in all the animals administered with the test substance,specific clinical symptoms or dead animals were not found, and in theweight change measurement, hematological tests, blood biochemical testsand autopsy findings, no change in toxicity was observed. Thus, it couldbe seen that the recombinant fusion protein of the present inventionshowed no toxicity in the rats even at a dose of 1 g/kg and that theminimum lethal dose (LD₅₀) upon oral administration thereof was 1 g/kgor more, suggesting that it is a safe substance.

The features and advantages of the present invention are as follows.

(a) The present invention provides a fusion protein comprising thetranscription modulation domain of the NF-κB subunit p65 and a proteintransduction domain, and the use thereof.

(b) The fusion protein of the present invention has the effects ofinhibiting the transcription of NF-κB and IL-2 by competitive inhibitionand inhibiting the LPS-induced secretion of inflammatory cytokines andalso inhibiting the production of IL-2, IFN-γ, IL-4, IL-17A and IL-10 insplenocytes, and thus can be effectively used as a composition for theprevention or treatment of a disease related to NF-κB overactivity.

Although the present disclosure has been described in detail withreference to the specific features, it will be apparent to those skilledin the art that this description is only of a preferred embodimentthereof, and does not limit the scope of the present invention. Thus,the substantial scope of the present invention will be defined by theappended claims and equivalents thereof.

REFERENCES

-   Park et al., Intranuclear interactomic inhibition of NF-kB    suppresses LPS-induced severe sepsis, Biochemical and Biophysical    Research Communications, 2015; 464:711-717.

1.-15. (canceled)
 16. A method for treating a disease related to NF-κBoveractivity, the composition comprising a fusion protein comprising atranscription modulation domain of NF-κB and a protein transductiondomain as an active ingredient, wherein the disease related to NF-κBoveractivity is septic shock or inflammatory disease, wherein theinflammatory disease is selected from the group consisting of allergicasthma, allergic nasitis, atopic dermatitis, systemic lupuserythematosus, rheumatoid arthritis, ulcerative colitis, dacryoadenitis,Alzheimer's disease, stroke, arteriosclerosis, vascular restenosis, typeI diabetes, type II diabetes, urticaria, conjunctivitis, psoriasis,systemic inflammatory response syndrome, polymyositis, dermatomyositis,polyarthritis nodosa, mixed connective tissue disease, Sjogren'ssyndrome, gout, Parkinson's disease, amyotrophic lateral sclerosis,diabetic retinopathy, multiple sclerosis, Crohn's disease, chronicthyroiditis, Celiac disease, radiation-induced disorders, hemangioma,reperfusion injury, and cardiac hypertrophy, wherein the fusion proteininhibits NF-κB transcription by competitive inhibition, wherein theNF-κB is RelA(p65).
 17. The method of claim 16, wherein thetranscription modulation domain of NF-κB has an amino acid sequence ofSEQ ID NO:
 1. 18. The method of claim 16, wherein the proteintransduction domain is selected from the group consisting of Hph-1,Mph-1, Sim-2, Tat, VP22, Antp (antennapedia), Pep-1 (peptide-1), PTD-5(protein transduction domain-5), 11R, 7R, and CTP (cytoplasmictransduction peptide).
 19. The method of claim 16, wherein the proteintransduction domain comprises an amino acid sequence of SEQ ID NO: 3.20. The method of claim 16, wherein the fusion protein comprises anamino acid sequence of SEQ ID NO: 5.